Chronic obstructive pulmonary disease (COPD), ranks as the third leading cause of death in the U.S. It is also strongly influenced by cigarette smoke (CS) and genetic predisposition. HHIP, encoding Hedgehog interacting protein, has consistently been associated with the susceptibility to COPD including airway remodeling and emphysema. However, the molecular mechanism underlying this association remains incompletely understood. Our published work has demonstrated that Hhip heterozygous mice (Hhip+/-) recapitulated multiple COPD pathological features including smoke- and age-related emphysema and airway remodeling. We also found that HHIP is highly expressed in Lgr6-expressing airway smooth muscle cells (ASMCs) and Lgr5- expressing alveolar mesenchymal cells; has reduced expression in COPD ASMCs that display a metabolic shift from oxidative phosphorylation to glycolysis associated with increased cell growth. Furthermore, alveolar fibroblasts-derived HHIP promotes proliferation of AT II (alveolar type II) cells in alveolar organoid co-culture model. These findings suggested that Hhip, the key genetic determinant for COPD, possibly modulates both airway remodeling and emphysema through complementarily intrinsic and extrinsic signaling in two major lung mesenchymal cell types: ASMCs and alveolar mesenchymal cells. In this current proposal, we aim to extend our previous studies by addressing two mechanistic questions related with HHIP: 1) How deficiency of Hhip in ASMCs promote airway remodeling by increasing airway thickening and cell hyperplasia through metabolic reprograming and 2) whether and how deficiency of Hhip in alveolar mesenchymal cells have impaired niche to support AT II cell generation. These questions will be addressed through the combinations of biochemical assays, lineage tracing, CRISPR-based genome editing and organoid co-culture models. In Aim1, we have identified a novel interaction between HHIP and PKM2 (pyruvate kinase isozyme M2), a rate-limiting enzyme in the last step for glycolysis. We will further characterize their interaction in AMSCs as well as determine impacts of HHIP on CS-induced airway remodeling. In Aim 2, we hypothesize that Hhip deficiency leads to impaired niche function in Lgr5+ alveolar mesenchymal cells that are important for AT II cells regeneration thereby determines emphysema susceptibility. To test this, we choose to conditionally deplete Hhip in Lgr5+ cells, a known alveolar mesenchymal cells marker followed by CS exposure and subsequent measurements on airspace size, the activity of the Wnt and Hedgehog pathway and proliferation and differentiation of both alveolar mesenchymal cells (Lgr5+) and AT II cells by complementary approaches in Aim 2.1. In Aim 2.2., we will use alveolar organoid co-culture model to dissect the mechanism by which Hhip determines niche function using either murine cells from Hhip deficient mice or from human primary fibroblasts edited by CRISPR/Cas-9 method targeting HHIP. Successful completion of this project will illuminate molecular insights into CS-induced COPD susceptibility determined by HHIP.